Inhibition of BAX promoter activity by LMP-1 is mediated by the NF-κB pathway. (A) Western blot for LMP-1 (top) and CAT assay (bottom) of HEK 293-T cells cotransfected with pBAX-CAT and with pHEBo (□) or pLMP-1 (▪) and with pcDNA-3 (columns 1-6) or pIκBα(S32/36A) (columns 7-8) and grown in the presence of 5 μg/mL CAPE (columns 3-4) or 100 μg/mL SN50 (columns 5-6). (B) Detection of p65 and p50 protein expression by Western blot (top) and CAT assay (bottom) of HEK 293-T cells cotransfected with pBAX-CAT and the vector pCMV-T (lane 1) or pCMV-T-p65 and pCMV-T-p50 in gradually changing ratios but at constant total amount (0.3 μg) (lanes 2-8) or with an IRF-1 expression plasmid (lane 9). ^*Significantly different from vector control (P < .05). (C) CAT assay as in panel B with pBAX-CAT replaced by pHIV-1-LTR-CAT. (D) Western blot for p65 and p50 expression (top) and CAT assay (bottom) of HEK 293-T cells cotransfected with pBAX-CAT and with vector pCMV-T (▪), equal amounts of pCMV-T-p65 and pCMV-T-p50 (□), or pCMV-T-p50 alone (▨) and with pcDNA-3 (columns 1-6) or pIκBα(S32/36A) (columns 7-9) and grown in the absence (columns 1-3, 7-9) or presence (columns 4-6) of 5 μg/mL CAPE. (E) Western blot for p65 (top) and p50 (bottom) in HEK 293-T cells cotransfected with pBAX-CAT and pCMV-T (left), pCMV-T-p65 (middle), or pCMV-T-p50 (right) expression plasmids and control siRNA (ctr-si), p65-siRNA (p65-si), or p50-siRNA (p50-si). (F) CAT assay of HEK 293-T cotransfected with pBAX-CAT (left) or pSV2-CAT (right) together with pHEBo (□) or pLMP-1 (▪) and with ctr-si, p65-si, or p65-si and p50-si. (G) CAT assay of 1852.4 cells grown 6 days in the absence (□) or presence (▪) of 2 μg/mL tetracycline, then transfected with pBAX-CAT and grown 3 days in the absence (left) or presence of 100 μg/mL SN50 (middle) or 1 μM Bay11-7082 (right). (A-D,F-G) CAT activity in control-transfected cells (first bar in each panel) was set to 100%. All experiments were performed in triplicate, and the mean and standard deviations are shown.